Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes. We are expressing full length NM2 in the baculovirus Sf9 system. We are studying their filament structure and how phosphorylation of both the heavy chain and light chain affects filament formation. We use a single filament motility assay system wherein we can image the movement of fluorescently labeled myosin filaments over actin filaments fixed to the surface. We are examining the copolymerization of NM2A and NM2B form co-polymers in vitro. We are collaborating with the Korn lab at NHLBI to study the effects of heavy chain phosphorylation on filament assembly. With Sarah Hitchcock-DiGregori we have examined the effect of various nonmuscle tropomyosin isoforms on the actin activated ATPase activity and in vitro motility of NM2A, NM2B and NM2C HMM isoforms and find that some TM isoforms activate one or more of the HMMs whereas some do not. We have shown that myosin 18A is a pseudo-enyzme that does not have an actin-activated MgATPase activity and does not move actin filaments in the in vitro motility assay. It associates weakly and nonproductively with actin. Although its tertiary structure strongly resembles that of myosin 2 class molecules including the presence of a long coiled-coil tail, it does not form filaments on its own. However, we show that it does co-polymerize with NM2A. Incorporation of myosin 18 into the NM2A filaments shortens its length and, if sufficiently high myosin 18 concentrations are used, the myosin 2A does not form filaments, but instead associates with a myosin 18A monomer to form an antiparallel heterodimer. We have expressed the S1, HMM and full length constructs for Drosophila nonmuscle myosin 2 and have shown that enzymatically the protein has a low duty ratio with phosphate release being rate limiting and that the enzymatic activity of the HMM and full length proteins require phosphorylation of the regulatory light chain. Electron microscopy shows that the myosin forms bipolar filaments of about 300 nm in length with about 14-16 myosins per filament.